Chen, Shun-Jia and Melnykov, Artem and Varshavsky, Alexander (2020) Evolution of Substrates and Components of the Pro/N-Degron Pathway. Biochemistry, 59 (4). pp. 582-593. ISSN 0006-2960. PMCID PMC7286083. doi:10.1021/acs.biochem.9b00953. https://resolver.caltech.edu/CaltechAUTHORS:20200103-103302869
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PDF (N-Degron pathways (Figure S1), amino acid sequence alignments (Figure S2), inhibition of translation by blasticidin in K. lactis (Figure S3), results of RT-qPCR assays (Figure S4), verification of K. lactis strains (Figure S5), K. lactis strains...)
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Abstract
Gid4, a subunit of the ubiquitin ligase GID, is the recognition component of the Pro/N-degron pathway. Gid4 targets proteins in particular through their N-terminal (Nt) proline (Pro) residue. In Saccharomyces cerevisiae and other Saccharomyces yeasts, the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 bear Nt-Pro and are conditionally destroyed by the Pro/N-degron pathway. However, in mammals and in many non-Saccharomyces yeasts, for example, in Kluyveromyces lactis, these enzymes lack Nt-Pro. We used K. lactis to explore evolution of the Pro/N-degron pathway. One question to be addressed was whether the presence of non-Pro Nt residues in K. lactis Fbp1, Icl1, and Mdh2 was accompanied, on evolutionary time scales (S. cerevisiae and K. lactis diverged ∼150 million years ago), by a changed specificity of the Gid4 N-recognin. We used yeast-based two-hybrid binding assays and protein-degradation assays to show that the non-Pro (Ala) Nt residue of K. lactis Fbp1 makes this enzyme long-lived in K. lactis. We also found that the replacement, through mutagenesis, of Nt-Ala and the next three residues of K. lactis Fbp1 with the four-residue Nt-PTLV sequence of S. cerevisiae Fbp1 sufficed to make the resulting “hybrid” Fbp1 a short-lived substrate of Gid4 in K. lactis. We consider a blend of quasi-neutral genetic drift and natural selection that can account for these and related results. To the best of our knowledge, this work is the first study of the ubiquitin system in K. lactis, including development of the first protein-degradation assay (based on the antibiotic blasticidin) suitable for use with this organism.
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Additional Information: | © 2020 American Chemical Society. Received: October 23, 2019; Revised: December 15, 2019; Published: January 2, 2020. The authors thank current and former members of the Varshavsky laboratory for their advice and assistance. Author Contributions: S.-J.C., A.M., and A.V. designed the experiments. S.-J.C. and A.M. performed the experiments. S.-J.C., A.M., and A.V. wrote the paper. All authors discussed the results and commented on the manuscript. This work was supported by National Institutes of Health Grants 1R01GM031530 and 1R01DK039520 (A.V.). The authors declare no competing financial interest. | |||||||||
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Issue or Number: | 4 | |||||||||
PubMed Central ID: | PMC7286083 | |||||||||
DOI: | 10.1021/acs.biochem.9b00953 | |||||||||
Record Number: | CaltechAUTHORS:20200103-103302869 | |||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20200103-103302869 | |||||||||
Official Citation: | Evolution of Substrates and Components of the Pro/N-Degron Pathway. Shun-Jia Chen, Artem Melnykov, and Alexander Varshavsky. Biochemistry 2020 59 (4), 582-593; DOI: 10.1021/acs.biochem.9b00953 | |||||||||
Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
ID Code: | 100496 | |||||||||
Collection: | CaltechAUTHORS | |||||||||
Deposited By: | Tony Diaz | |||||||||
Deposited On: | 05 Jan 2020 03:37 | |||||||||
Last Modified: | 16 Nov 2021 17:54 |
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